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Characterization of
Decellularized Extracellular
Matrix for Clinical Use
Nikhil Gheewala, PhD
Principal Scientist, ACell
CASSS - Thursday, April 5th, 2018
Extracellular Matrix
Cells
Collagens
Elastin
Proteoglycans
Secreted and Bound Components
Decellularization (Ideal)
Collagens
Elastin
Proteoglycans
Secreted and Bound Components
Goal: Maximize ECM retention and cell removal while minimizing ECM damage
Decellularization Modifies the
Immune Response
Without decellularization, implanted xenogeneic and most
allogeneic tissues prompt a classic inflammatory (M1 macrophage)
response, often resulting in encapsulation or scar formation
More effective decellularization can result in:
Increased “anti-inflammatory” (M2 macrophage) response
Reduced encapsulation and scar formation
Constructive remodeling toward site-appropriate tissue
Brown 2009, Biomaterials;
Keane 2012, Biomaterials;
Current Clinical Uses of ACell’s
decellularized Urinary Bladder Matrix
(UBM) – “MatriStem UBM”
Regulated as Medical Devices
Wounds
Chronic Wounds
Burns
Pressure Ulcers
Diabetic Ulcers
Surgical
Surgical reinforcement
Plastic and reconstructive
surgery
*Preclinical data
• Host remodeling response includes
• Angiogenesis1 and innervation2
• Modulation of the inflammatory response1
• Participation of autologous progenitor cells3,6
• Resistance to infection5
• Host deposition of functional, site-appropriate tissue 4,6,7
• Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling by native cells
1. Brown et al., Acta Biomater 2012
2. Agrawal et al., J Tissue Eng
Regen Med 2009
3. Agrawal et al., PNAS 2010
4. Sicari et al., Sci Transl Med
2014
5. Medberry et al., J Surg Res
2012
6. Gilbert et al., J Surg Res 2008
7. Remlinger, et al.,
Organogenesis, 2012
Urinary Bladder Matrix (UBM)
Testing and Characterization
With tissue-derived medical devices, ACell can use a
combination of physical and analytical tests
Physical Testing
• Tensile Strength
• Suture Retention
Strength
• Calorimetry (collagen
thermal stability)
• Ease of Handling
Analytical Quantification
• Moisture Content
• DNA
• Phospholipids
• Collagens
• Glycosaminoglycans
• bFGF
EXTRACTION METHODS
Some material components can be extracted without requiring enzymatic
digestion of the tissue macrostructure, using appropriate buffers and
micronizing/homogenization methods
Phospholipid Quantification
• Phospholipid content is indicative of the level of remaining cellular and
intracellular membrane components
• Measurable in solution using BioAssay Systems’ EnzyChrom™ Phospholipid
Assay Kit
• Need to be able to extract into solution a repeatably high yield of phospholipids
• Initial suggestion from BioAssay Systems was to use Triton X-100, also used
as the provided “assay buffer”
Phospholipid Buffers
Buffers tested:
Triton X-100, baseline
Triton X-100 + 50mM Tris buffer
Methanol
• Dried after extraction, then reconstituted in Triton X-100 +
50mM Tris
Test Articles:
Micronized (milled) UBM samples, non-sterile
Phospholipid Extraction Buffers
100.0%
99.6%
110.1%
1.3% 1.8% -0.9%
-20%
0%
20%
40%
60%
80%
100%
120%
140%
Triton X-100 Triton X-100 +50mM Tris
Methanol
Yie
ld
(% o
f Tri
ton
X-1
00 E
xtr
ac
tio
n)
Extraction Methods
Normalized Extraction Yields
1st extraction
2nd extraction
MultiPlex ELISA
Informational multiplex assays are occasionally used for a
variety of analytes
Each analyte is not tested enough to justify full
development
We’d like to have a “one-size fits all” approach to allow
gross evaluation of a number of analytes
Do we digest the material or homogenize in MPER
(mammalian protein extraction reagent)?
MultiPlex ELISA Digestion or
Extraction
• Results excluded when non-detectable
• Homogenate extract, with MPER, is generally more effective
0.1
1
10
100
1000
10000
100000
IL-1
raIL
-4IL
-7IL
-18
FG
F-1
FG
F-2
FG
F-2
EG
FV
EG
FV
EG
F-a
PD
GF
PLG
FM
CP
-1M
CP
-3M
DC
FIT
-3L
Fra
cta
lkin
eR
AN
TE
S
Mass c
on
ten
t (n
orm
alized
to
IL
-1ra
)
UBM MultiPlex ELISA
Pepsin tissue digest
Homogenate extract
DIALYSIS AND/OR DILUTION
After extraction or sample digestion, do we need to remove potential
contaminants?
bFGF Quantification
bFGF: basic Fibroblast Growth Factor (FGF-2)
Can be measured as a representative of a large number of
heparin-binding proteins
Not known to affect product performance, but we often receive
inquiries from physicians about GF content
Measurable in solution using a number of available human bFGF
ELISA kits
We need a sample solution that won’t interfere with specific
antibody binding
bFGF Extraction Buffer and
Dialysis
Buffers tested:
Medium 106, with protease inhibitors
Urea, with heparin, tris, and protease inhibitors
Dialysis
Each buffer tested with or without 2x 24hr dialysis
Test Articles:
Micronized (milled) UBM samples, non-sterile
bFGF Extraction Buffer and
Dialysis
0
0.5
1
1.5
2
2.5
3
Medium 106 Medium 106 w/Dialysis
Urea Urea w/ Dialysis
bF
GF
Co
nte
nt
(No
rma
lize
d t
o M
ed
ium
10
6)
Treatment Group
Urea Extraction Yield Confirmation
• Spike recovery experiments performed by spiking bFGF either into
the powder, followed by drying, or into the extract
0
0.5
1
1.5
2
2.5
3
Normal Powder Spike Extract Spike
Measu
red
bF
GF
Co
nte
nt
(no
rma
lize
d t
o N
orm
al)
Spike Recovery of bFGF in UBM
Calculated Spike
Normal Pre-spike Content
Measured Values
dsDNA Quantification
• DNA content is indicative of the level of remaining nuclear (or
mitochondrial) components
In addition, DNA specifically can negatively influence an immune response
• Measurable in solution using Quant-IT Picogreen
• Tissue digestion is required to release DNA into solution, creating high
levels of turbidity
• Dilution is commonly used, but to what extent for our samples?
Dilutions of DNA assay digest
Test Articles:
Micronized (milled) UBM samples, non-sterile
Digested by Proteinase-K enzyme in lysis buffer
Dilutions
Single-step dilutions 1:10 to 1:100
Two-step dilutions 1:200 to 1:1000
Dilutions of DNA assay digest
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200
Ca
lcu
late
d D
NA
Co
nte
nt
(no
rma
lize
d t
o 1
:10
0)
Dilution Factor
1:100 Dilution – No change to procedure
In Conclusion
• Testing of dECM products largely focuses on materials
testing and “representative” compositional assays
• With soft tissue materials, our unique challenges generally
are related to the sample preparation – bringing our
analytes into a stable solution that won’t interfere with
commercially-available assay
• Like most testing, different types of analytes are suited to
different methods, and the simplest option is often the
most effective
Acknowledgements
Alphaba Sacko (Phospholipid)
Kevin Joye (DNA)
Jaci Miller (bFGF)
Penelope Morel, U Pitt (Multiplex ELISA)